Fatty acids are organic acids having a hydrocarbon chain of from about 4 to 24 carbons. Many different kinds of fatty acids are known which differ from each other in chain length, and in the presence, number and position of double bonds. In cells, fatty acids typically exist in covalently bound forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules is often depicted by the formula CX:Y, where "X" indicates number of carbons and "Y" indicates number of double bands. As the carbon chain of fatty acyl molecules always contains an even number of carbons, the formula "C.sub.2x " may also be used to represent carbon chain length.
Fatty acyl groups are major components of many lipids, and their long, non-polar hydrocarbon chain is responsible for the water-insoluble nature of these lipid molecules. The type of covalent linkage of the fatty acyl group to other factors can vary. For example, in biosynthetic reactions they may be covalently bound via a thioester linkage to an acyl carrier protein (ACP) or to CoenzymeA (CoA), depending on the particular enzymatic reaction. In waxes, fatty acyl groups are linked to fatty alcohols via an ester linkage, and triacylglycerols have three fatty acyl groups linked to a glycerol molecule via an ester linkage.
Many plants have been studied which store lipid as triacylglycerols composed primarily of long chain (having 16 or 18 carbons) fatty acyl groups. Very long chain (having 20-24 carbons) monounsaturated fatty acyl groups are formed by an acyl-CoA elongation pathway from C18:1 and are found in many plant seeds, notably members of the Crucifereae family. The desert shrub, Simmondsia chinensis, better known as jojoba, is unique among higher plants (seed-bearing plants) in its ability to produce and store large amounts of liquid wax as the major component of its seed storage lipid. These simple wax compounds are oxygen esters of very long-chain monoenoic fatty acyl groups and alcohols.
Other types of waxes are formed by some plant species. The synthesis of plant epidermal, or cuticular wax, as well as wax synthesis by bacteria, such as Acinetobacter (Fixter et al. (1986) J. Gen. Microbiol. 132:3147-3157) and Micrococcus (Lloyd (1987) Microbios 52:29-37), and by the unicellular green algae, Euglena, are well known. However., the composition and biosynthetic pathway of these waxes differs from the jojoba seed wax.
In the formation of Euglena storage wax for instance, it has been demonstrated that the alcohol portion is formed by an NADH-dependent reduction of a fatty acyl compound catalyzed by a fatty acyl-CoA reductase. In jojoba seeds, the reaction is NADPH-dependent. It has been postulated that the reduction of a very long chain fatty acyl-CoA to the corresponding alcohol is dependent upon a single enzyme whose activity has been observed in crude extracts from developing jojoba seeds (Pollard et al. (1979) Lipids 14:651-662; Wu et al. (1981) Lipids 16:897-902). Also, by comparison, for the formation of plant cuticular waxes, a two step process has been reported (Kolattukudy (1980) in The Biochemistry of Plants (Stumpf, P. K. and Conn, E. E., eds.) Vol.4, p. 571-645). The fatty acyl-CoA is converted to a free aldehyde by the action of an NADH-dependent reductase and the alcohol is subsequently formed by the action of an NADPH-dependent fatty aldehyde reductase.
Further characterization of the enzymes responsible for formation of wax esters in plants has been hindered by the association of these factors with a floating wax pad which is formed upon differential centrifugation of a cell-free homogenate. It is desirable, therefore, for further study of plant fatty acyl reductase proteins to devise a purification protocol whereby these proteins may be separated from the wax pad, especially with a goal to provide a solubilized protein preparation. By establishing these methods, sufficient amounts of plant fatty acyl reductase protein may be obtained, the amino acid sequence of the protein may be determined and/or antibodies specific for the fatty acyl reductase may be obtained. The resulting amino acid sequences may be useful in polymerase chain reaction (PCR) techniques or for screening cDNA or genomic libraries. Alternatively, antibodies may be used for screening expression libraries to identify clones expressing fatty acyl reductase protein. Clones obtained in this manner can be analyzed so that the nucleic acid sequences corresponding to plant fatty acyl reductase are identified.
Relevant Literature
Cell-free homogenates from developing jojoba embryos were reported to have NADPH-dependent fatty acyl-CoA reductase activity. The activity was associated with a floating wax pad which formed upon differential centrifugation (Pollard et al. (1979) supra; Wu et al. (1981) supra).
Conservation of functional residues in known dinucleotide binding folds of several reductase proteins is presented by Karplus et al. (Science (1991) 251:60-66).
Solubilization of a multienzyme complex from Euglena gracilis having fatty acyl-CoA reductase activity is reported by Wildner and Hallick (Abstract from The Southwest Consortium Fourth Annual Meeting, Feb. 7, 1989, Riverside, Calif.).
3000-fold purification of jojoba reductase protein is reported by Pushnik et al. (Abstract from The Southwest Consortium Fourth Annual Meeting, Feb. 7, 1989, Riverside, Calif.).